This invention was made at least in part with funds from the Federal government, and the government therefore may have certain rights in the invention.
The field of the invention relates to cancer therapy; human immunodeficiency virus; a recombinant macrophage; methods of diagnosis of macrophage involvement in cancer development; kits for use in diagnosis; and methods of treatment for cancer involving macrophage-induced tissue growth.
Of the approximately 36,000 new cases of lymphoma diagnosed in the United States in 1992, between 8 and 27-o are estimated to have occurred in HIV-infected individuals (Gail, M. H. et al., J Natl Can Int (1991) 83:695-701). Thus, HIV-related lymphoma represents a major clinical problem for physicians involved in the care of HIV-infected individuals.
The biology of AIDS lymphoma is controversial and appears complex. Early in the AIDS epidemic high grade non-Hodgkin""s lymphoma (NHL) began to appear in individuals at risk for the development of AIDS (Ziegler, J. et al., N Eng J Med (1984) 311:565-570). However, in the past several years, the incidence of NHL in HIV-infected individuals has increased (Harnly, M. E. et al., Am J. Epi (1988) 128(2):261-267; Levine, A. et al., Ann Intern Med (1984) 100:7-13). It is clear that as the AIDS epidemic expands, non-Hodgkin""s lymphoma will become a continually more important health problem in HIV-infected individuals. As treatment for the underlying HIV disease becomes more successful and as patients survive for longer periods of time in-the absence of opportunistic infections, more cases of lymphoma will probably appear in this patient population.
The non-Hodgkin""s lymphomas that develop in HIV-1 infected individuals fall into two main subcategories: the large cell lymphomas and the small non-cleaved cell Burkitt""s lymphomas (Ziegler, J. et al., 1984, supra; Knowles, D.M. et al., Blood (1989) 73:792-799; Bermudez, M. et al.; Am J Med (1989) 86:71-76; Gill, P. et al., J Clin Oncol (1987) 5:1322-1328; Kaplan, L. D. et al, JAMA (1989) 261:719-724; Knowles, D. M. et al., Ann Intern Med (1988) 108:744-753; Lowenthal, D. A. et al., Cancer (1988) 61:2325-2337). Both major classes of lymphoma are high grade 5neoplasms and are predominantly of B-cell origin (Ziegler, J. et al., 1984, supra.; Subar, M. et al., Blood (1988) 72:667-671.); however, T-cell lymphomas may also be increasing in frequency (Presant, C. A. et al., Cancer (1987) 60:1459-1461; Nasr, S. et al., Cancer (1988) 61:947-951; Herndier, B. et al., VII Intl Conference of Acquired Immunodeficiency Syndrome (AIDS), Florence, Italy, Jun.16-21, 1991). In HIV disease lymphomas tend to be diffusely aggressive, with approximately 90% originating from B-cells and 5-10% derived from T-cells. Approximately one-half of the large cell lymphomas are herein termed xe2x80x9cmixed immunophenotypexe2x80x9d lymphomas as they contain a mixture of B-cells, T-cells, and macrophages. AIDS-associated non-Hodgkin""s lymphomas are commonly characterized by their very high rates of extranodal (85-97%) (Kaplan, L. D. et al., JAMA (1989) 261:719-724; Burkes, R. L. et al., Arch Intern Med (1986) 146:913-915; Balasubramanyam, A. et al., Chest (1986) 90:243-246; Guarner, J. et al., Arch Pathol Lab Med (1987) 111:254-256; Kaplan, L. et al., Ann Intern Med (1989) 110:162; Friedman, S. L., Gastroenterol Clin North Am (1988) 17:465-486) and central nervous system involvement (35k) (Baumgartner, J. et al., J Neurosurc (1990) 73:206-211; Formenti, S.C. et al., Cancer (1989) 63:1101-1107; Ciricillo, S. et al., J Neurosurq (1990) 73:720-724), as well as their poor response to current chemotherapy protocols (Kaplan, L. D. et al., (1989) supra; Bermudez, M. et al., Am J Med (1989) 86:71-76; Gill, P. et al., J Clin Oncol (1987) 5:1322-1328; Urba, W. et al., Journal of the National Cancer Institute (1990) 10:29-37; Kaplan, L. D. et al., JCO (1991) 9(6):929-940).
Lymphomas, in general, are a heterogeneous group of malignancies. Their biologic behavior ranges from indolent, requiring no therapy, to aggressive malignancies with few long-term survivors. The behavior of lymphoma is influenced by the immune status of the host. The risk of B-cell lymphoma is dramatically increased in individuals with defects of cell-mediated immunity. The best characterized of these groups is immunosuppressed allograft recipients, whose risk of developing lymphoma is between 50 and 60 times that of the general population. These individuals develop a spectrum of lymphoproliferative diseases ranging from typical monoclonal immunoblastic lymphoma to an aggressive form of polyclonal lymphoproliferative disease (Frizzera, G. et al., Cancer Res (1981) 41:4262-4279; Hanto, D. W. et al., Cancer Res (1981) 41:4253-4261; Hanto, D. W. et al., Ann Surg (1983) 198:356-369) often associated with Epstein Barr Virus (Hanto, D. W. et al., (1981) supra; Penn, I., Transplant Proc (1983) 15 (suppl 1):S2790-S2797; Shearer, W. T. et al., N Engl J Med (1985) 312:1151-1159) infection. Clinically, lymphoma in these individuals presents aggressively at extranodal sites indicating a common feature between HIV-associated lymphomas and the molecular and clinical characteristics of the allograft-associated lymphomas.
The primary means of HIV lymphoma diagnosis remains microscopic examination of hematoxylin and eosin-stained sections from formalin-fixed tissue. Over time, pathologists have used clinical presentations, autopsy follow-up, and trial and error to develop histologic methods a of diagnosing and categorizing cancer. Missing a histologic diagnosis of cancer or xe2x80x98over-callingxe2x80x99 a cancer and subjecting a patient to cancer therapy are sufficient incentives for providing accurate diagnosis. Traditional histologic methods can be enhanced by phenotypic and, particularly, genotypic analyses of lymphomas where the discerned molecular changes of the affected tissue point to an alternative form of treatment.
The invention. relates to a method of diagnosing clonal macrophage involvement in HIV-associated and non-HIV-associated lymphomas or other cancers using genotypic analysis as well as a kit for such diagnostic method. The invention also relates to a method of treating macrophage-induced cancer. The invention also relates to a recombinant macrophage useful in vitro and in vivo methods of screening for therapeutic agents useful in treating macrophage-induced cancer.
The discovery that HIV lymphomas are frequently associated with clonal macrophage involvement and that the macrophage has HIV DNA integrated upstream of a known oncogene, c-fes (c-fes/fps), is disclosed. As described in detail below, macrophage clonality is associated with many HIV-related lymphomas. Macrophage clonality can be associated with non-HIV-related lymphomas as well. Expansion of macrophages may enhance growth of surrounding tissue by secretion of cytokines; the cytokine Interleukin-6 has been shown to cause growth of myeloma and hybridoma cells (Woodruff, C. et al., DNA and Cell Bioloqy 11:587-592). Diagnosis of macrophage clonality and treatment targeting macrophages offers a new direction in cancer therapy. Disclosed are diagnostic methods and kits as well as therapeutic methods useful in the battle to overcome clonal macrophage-induced HIV lymphomas and clonal macrophage-induced cancers in general.
Accordingly, in one aspect, the invention features a method of diagnosing the presence of clonally expanded macrophages in a suspected cancerous tissue of a mammal by first obtaining a sample of the tissue suspected of being cancerous followed by isolation of DNA from the tissue by standard techniques known to those skilled in the art of molecular biology. The presence of clonal DNA in the isolated DNA is determined by standard techniques including but not limited to HIV integration site analysis by IPCR (Shiramizu, B. et al., Cancer Res (1994) 54:2069-2072); RFLP (Restriction Fragment Length Polymorphism) analysis of genomic sequences near common sites of viral (e.g., HIV) integration; or immunoglobulin (Ig) gene rearrangement analysis (Levy, R. et al., J Exy Med (1977) 145:1014-1028). Preferentially, this analysis includes mixing known quantities of control monoclonal DNA and control polyclonal DNA such that, following Southern analysis, the DNA band intensity of a 5% monoclonal mixture is determined and used as a standard against which the clonality of the test DNA is compared. Using this technique, monoclonal tumors are those defined as having more than a 5% monoclonal DNA component. Preferably, the technique of inverse polymerase chain reaction (IPCR) used to determine the clonality of a tissue sample containing integrated HIV is a valuable diagnostic tool and is defined in detail below.
The invention also features a kit for the diagnosis of macrophage-induced pre-cancerous and cancerous tissue as tissue containing greater than 5% monoclonal DNA from macrophages. The kit of the invention features nucleic acid primers for amplifying DNA of an HIV-containing cell (e.g., a macrophage). The nucleic acid primers for amplification of HIV-containing DNA preferentially hybridize to regions of the 5xe2x80x2 and /or 3xe2x80x2 long terminal repeats (LTRs) such that DNA synthesis is primed in opposite directions for IPCR analysis. The kit also includes a labeled (e.g., radiolabeled; biotinylated; or other standard label) probe for the RFLP analysis of genomic DNA for clonality where priming of HIV sequences is not desirable (e.g., in non-HIV-related cancers). Such a probe hybridizes to a genetic locus which is a common site of viral integration leading to cellular proliferation.
The diagnostic method of the invention features comparing the clonality of test DNA to control HIV-containing DNA to indicate involvement of HIV-containing clonal cells (e.g., clonal macrophage) in the tissue morphology. The involvement of macrophages is determined by initial sorting into cellular subpopulations. (by fluorescence activated cell sorting (FACS) technique or macrophage adherence to glass or plastic; or other technique well known in the art) followed by DNA analysis. Alternatively, analysis of DNA from a mixed cellular population is performed followed by dual staining or in situ hybridization of a tissue preparation to correlate clonal HIV with the macrophages.
The diagnostic method of the invention also features analysis of the clonality of non-HIV-associated macrophages. The method involves isolating test DNA from a mixture of cells or a pre-sorted subpopulation of macrophages from pre-cancerous or cancerous tissue followed by RFLP analysis of the test DNA. RFLP analysis of the invention utilizes a nucleic acid probe that hybridizes to a genomic sequence encoding a common viral (e.g., HIV) integration site associated with cellular proliferation upon viral integration. For example, HIV integration into genomic DNA in the z exon of the fur gene has been observed in both T-cells (Shiramizu, B., et al. (1994), supra) and in macrophages as described herein.
Using the diagnostic method of the invention, the absence of HIV clonality can indicate a non-HIV-associated clonal macrophage involvement in the tissue morphology. The absence of both HIV and macrophage involvement is also discernable using the kit of the invention. The diagnosis of macrophage involvement provides the clinician with powerful information for designing an appropriate treatment regimen for the patient.
The invention features a macrophage having integrated in its genome a transcriptional/translational controlling sequence operably attached to a cell proliferation gene such that expression of the cell proliferation gene results in (expansion) of the macrophage. Cell proliferation can be the result of substantially increased expression of a cytokine gene or other cell proliferation gene such as an oncogene. For example, integration into the z exon of the macrophage fur gene of a strong promoter and/or enhancer element (e.g., an HIV genome; a defective HIV genome wherein the enhancer region of an HIV 3xe2x80x2 LTR is functional; the enhancer region of an HIV 3xe2x80x2 LTR; or other promoter or enhancer element capable of substantially increasing expression of a nearby gene (whereby nearby is meant within about 12 kb, preferably about 10 kb, or more preferably about 5 kb of the transcriptional start site) such that expression of the downstream c-fes/fps gene is substantially increased relative to c-fes/fps expression in a macrophage having no promoter or enhancer element integrated within it. Integration of a DNA sequence encoding a strong promoter and (optionally an enhancer element) operably attached to a growth promoting cell proliferation gene into the macrophage genome is accomplished by standard molecular biology techniques (e.g., delivery of the transfecting DNA in liposomes) (see e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Green Publishing Associates, Cold Spring Harbor Laboratory Press, 1989; and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, new York, 1989). Recombinant macrophages are screened for increased c-fes/fps expression by techniques described herein. Alternatively, the transcriptional/translational controlling regions are fused to a cell proliferation gene on a DNA expression cassette which is integrated into the macrophage genome. Expression of the cell proliferation gene of the cassette also results in cell proliferation. Recombinant macrophages are also screened for the ability to enhance cell proliferation relative to non-recombinant macrophages when introduced into a population of B- and/or T-cells.
Macrophages which express increased levels of cell proliferation controlling gene such as the oncogene c-fes/fps are useful for screening therapeutic agents in vitro for the ability to inhibit growth of such macrophages. The screening method of the invention provides cultured recombinant macrophages expressing a substantially increased level of c-fes/fps in an appropriate culture medium. To the cultured recombinant macrophages is added a therapeutic agent in an appropriate formulation for delivery of the therapeutic agent to the recombinant macrophage. The effect of administration of the agent is monitored as reduced cell growth, reduced cell viability, reduced c-fes/fps expression, and/or reduced cytokine expression. Methods of monitoring cell growth and viability are well known in the art. Methods of monitoring c-fes/fps and cytokine expression are described herein. Therapeutic agents which cause reduction in the monitored phenotypes are selected as candidate compounds or formulations for treatment of macrophage-induced cancers such as those listed in Table II.
The invention features a method of screening candidate therapeutic agents in vivo for the ability to substantially eliminate a recombinant macrophage (e.g., a clonally expanded macrophage having a transcription controlling sequence integrated for the increased expression of a cell proliferation gene) from the host. The recombinant macrophage is injected into the appropriate tissue of the host (e.g., the spleen, the peripheral blood, the skin, or the bone marrow) and growth of a tumor containing the recombinant macrophage is monitored. To the host is then administered a candidate therapeutic agent in a formulation for preferential uptake by a macrophage, wherein the administering is performed at a dose, an interval, and for a duration that is sufficient to substantially eliminate the recombinant macrophage from the host.
Another feature of the invention is screening or a candidate therapeutic agent for treatment of cancers induced by the presence of clonally expanded macrophages. The screening method involves transplantation of mammalian tumor tissue containing clonally expanded macrophages (such as a recombinant macrophage which contains a growth promoting cell proliferation gene operably attached to a functional transcriptional controlling region such as a promoter/enhancer region) into a mammal (such as a severe combined immumodeficiency (SCID) mouse). A cancer is allowed to develop in the mammal containing the implanted tissue. A pharmaceutically effective amount of a candidate therapeutic agent is administered to the mammal to determine if administration of the agent results in reduction in the size of the tumor, reduction in clonal macrophage viability, or reduction in clonal macrophage proliferation.
Another feature of the invention is a method of treatment of clonal macrophage-involved cancer by first determining the presence of a clonally expanded macrophage in the cancer. Second, the macrophage is substantially eliminated from the cancerous tissue by administering to the mammal (preferably a human) harboring the cancerous tissue, a therapeutic agent that is in a formulation for preferential uptake by a macrophage. The administering is performed at a dose, at an interval and for a duration that is sufficient to substantially eliminate the macrophages (including the clonally expanded macrophages) from the mammal followed by discontinuation of the administering of the therapeutic agent such that the macrophage population of the mammal is regenerated.
Another feature of the invention is the treatment method described above wherein the therapeutic agent is DNA encoding antisense MRNA of a cell proliferation gene. The target cell proliferation gene is one positioned near (i.e., within 12 kb, preferably within 5 kb in distance from) a common site of HIV integration wherein the common site of HIV integration is shown by the diagnostic method of the invention to be clonally mutated. Introduction of the antisense-encoding DNA into the macrophage genome and expression of the antisense MRNA reduces expression of the cell proliferation gene.
Another feature of the invention is a therapeutic agent comprising DNA encoding a mutant form of a cell proliferation gene such that recombination into the endogenous cell proliferation gene of the macrophage genome results in the mutation being incorporated into the genome. The mutation of the cell proliferation gene is designed such that gene expression is substantially eliminated or that the expressed gene produces a substantially nonfunctional gene product.
Those skilled in the field of molecular biology will understand that any of a variety of conventional gene transfer methods may be used for introducing genes into cells. For example, physical methods for the introduction of DNA into cells include microinjection (see, e.g., Capecchi et al., Cell 22:479, 1980), electroporation (see, e.g., Reiss et al., Biochem. Biophys. Res. Commun. 137:244, 1986) and other standard methods. Chemical methods such as coprecipitation with calcium phosphate and incorporation of DNA into liposomes also have been used to introduce DNA into mammalian cells, particularly macrophages as described herein. Finally, delivery of nucleic acids into mammalian cells can be executed through the use of viral vectors, in particular those derived from murine and avian retroviruses (see, e.g., Gluzman et al., Viral Vectors, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988).
The invention can utilize a commercially available cytotoxic therapeutic agent such as trichosanthin (Lifson, J. D. et al., USPN 4,795,739; USPN 4,869,903; McGrath, M. S. et al., PNAS (1989) 86:2844-2848), alpha- or beta-momorcharin (Lifson, J. D., et al., supra), other single-chain ribosome inactivating protein or other cytotoxic agent well known in the art of HIV inhibition or cancer therapy. The cytotoxin may comprise multiple agents such that the combined actions of the agents results in cytotoxicity.
Trichosanthin is a plant protein obtained from the Trichosanthes kirilowii root tuber. It has homology to the amino acid sequence of Ricin A chain and may have ribosome-inhibitory properties similar to ricin and various single-chain ribosome-inhibitor proteins, such as momorcharin, pokeweed anti-viral protein (PAP), wheat germ inhibitor, and gelonin (Xuejun) (Lifson, J. D., et al., supra). Trichosanthin and momorcharin inhibit expression of HIV antigens in human blood cells including macrophages (Lifson, J. D., et al., supra).
Examples of other cytotoxic or proliferation-modulating agents include daunomycin, mitomycin C, daunorubicin, doxorubicin, 5-FU, cytosine arabinoside, colchicine, cytochalasin B, bleomycin, vincristine, vinblastine, methotrexate or the like. Also of interest are toxic agents which may be derived from microorganism or plant sources. Examples include the toxic subunits of naturally occurring toxins such as ricin, abrin, diphtheria toxin, saporin, and the like. Illustrative toxic subunits include the A chains of diphtheria toxin, enzymatically active proteolytic fragments from Pseudomonas aeruginosa exotoxin-A, ricin A-chain, abrin A-chain, modeccin A-chain, and proteins having similar activity found in various plants such as the plants Gelonium multiflorum, Phytolacca Americana, Croton, Tiglium, Jatropha, Curcas, Momordic, Charantia, Reachan, the toxin saporin from Saponaria officinalis (Thorpe et al. J. National Cancer Institute (1985) 75:151), the Chinese cucumber toxin, trichosanthin (Yeung et al. Intl. J. of Peptide Protein Res. (1985) 27:325-333). Mutant species of the toxins also may be used, for example, CRM 45 (Boquet et al. Proc. Natl. Acad. Sci. USA (1976) 73:4449-4453).
The cancer treatment method of the invention features a liposome preparation containing within the liposome a macrophage-specific cytotoxin or a broad-spectrum cytotoxic agent. Such cytotoxin-containing liposome is prepared to be a size appropriate for the uptake of the cytotoxin-containing liposome preferentially by a macrophage. Targeting of the cytotoxin-containing liposome to a macrophage provides specificity of delivery and increased uptake. Targeting is accomplished by incorporation or attachment of a macrophage-specific antibody such as anti-CD14 to the liposome. Appropriate lipids and other agents and methods for the preparation of therapeutic liposomes are well known in the art (see e.g., Martin, F. J. and Papahadjopoulos, D., J. Biol. Chem. 257:286-288, (1982); Szoka, F. and Papahadjopoulos, D., Ann. Rev. Biophys. Bioeng. 9, 467-508, (1980); and Ostro, M. J. (ed) Liposomes From Biophysics to Theraueutics, Marcel Dekker, Inc., New York, 1987).
The cancer treatment method of the invention additionally features an anti-macrophage cell surface antibody covalently attached to a cytotoxic agent such that the cytotoxic agent is preferentially taken up by a macrophage following attachment of the cell surface antibody-cytotoxin complex to a macrophage cell surface marker. Uptake of the complex is by a phagocytic process normal to the macrophage.
The proliferation-modulating agent and the targeting agent which provides binding to the extracellular matrix can be linked, usually by a bond which is cleavable, either intra- or extracellularly by reduction hydrolysis enzymatically, or by a bond which is acid labile. The type of linkage used depends upon a number of factors, but particularly the nature of the proliferation-modulating agent. For example, where the agent is one which must be internalized by a cell to have an effect, such as a toxin molecule or toxin A chain, it is preferable that the linkage to the targeting moiety can be cleaved. The targeting moiety can be linked to a drug either directly or indirectly by carrier molecules such as serum albumin (particularly human serum albumin), polyaminoacids, dextran, and the like, by methods well known to those skilled in the art. The use of a carrier molecule permits binding of multiple molecules of the proliferation-modulating agent to the linker molecule, preferably 10 to 30 molecules per molecule of carrier molecule for an antiproliferative compound, or 1 to 2 molecules per molecule of carrier molecule for a toxin molecule.
The type of linkage used can also be dictated by the cell type which is the ultimate goal of the proliferation modulating activity thus preferably the linkage is a Ph labile or acid labile link between the targeting moiety and the proliferation-modulating moiety in cases where the-cells have phagocytotic properties, for example, fibroblasts and macrophages. Also of interest are peptide linkages which would be susceptible to hydrolysis by enzymes which may also be present in the extracellular matrix. Convenient linkages thus include disulfides, imides, hydrazones, amides and the like.
The cancer treatment method of the invention additionally features the complementary treatment of the non-macrophage cells of the tumor by conventional cancer therapy methods such that both the clonally expanded macrophage of the tumor and the non-macrophage cells of the tumor are each substantially ablated.
By xe2x80x9cclonal macrophagexe2x80x9d or xe2x80x9cclonally expanded macrophagenxe2x80x9d is meant identical copies of a macrophage arising from a single progenitor macrophage.
By xe2x80x9cclonality of DNAxe2x80x9d is meant the degree to which DNA isolated from a cell population is the same as determined by the pattern of discernable DNA characteristics such as RFLP (restriction fragment length polymorphism), gene rearrangement by Southern analysis, IPCR, or other techniques known to those skilled in the art. Accordingly, a tissue sample is designated as having a monoclonal macrophage component if practice of the diagnostic method of the invention results in a distinct HIV-specific and/or macrophage-specific DNA band pattern of equal or greater intensity than a 5% control sample.
By xe2x80x9c5% control samplexe2x80x9d is meant a sample of known polyclonal DNA to which has been added a sample of known monoclonal DNA to constitute 5% of the total DNA.
By xe2x80x9cpromoterxe2x80x9d is meant minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific or inducible by external signals or agents; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 regions of the native gene.
By xe2x80x9cintegrated into the genomexe2x80x9d is meant exogenous DNA (such as an expression cassette or viral sequence) that is linearly incorporated within the genomic DNA chain of a cell (such as a macrophage) and is connected at each of its termini to the genomic DNA. Exogenous DNA encoding an expression cassette is integrated such that functional RNA or proteins are produced by the encoded sequences. An exogenous viral sequence is integrated such that expression of nearby endogenous
By xe2x80x9cenhancerxe2x80x9d is meant minimal sequence sufficient to enhance transcription substantially above wild type levels. Also included in the invention are those enhancer elements which are sufficient to enhance promoter-dependent gene expression controllable for cell-type specific or inducible by external signals or agents; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 regions of the native gene.
By xe2x80x9cpharmaceutically active amountxe2x80x9d is meant an amount of an agent (such as a therapeutic agent) that when administered to a cell or a mammal results in a desired physiological effect (such as cytotoxicity, reduced viability, or substantially reduced proliferation).
By xe2x80x9csubstantial increasexe2x80x9d or xe2x80x9csubstantially abovexe2x80x9d is meant an increase in expression, transcription or other process above the wild type level of that process where the increase is at least approximately 50% above wild type.
By xe2x80x9csubstantial decreasexe2x80x9d or xe2x80x9csubstantial reductionxe2x80x9d is meant a decrease or reduction in expression, transcription, or a measurable phenotypic characteristic that is approximately 80% of the wild type level, preferably reduced to approximately 50% of the wild type level, or more preferably reduced to approximately 10% or less of the wild type level.
By xe2x80x9csubstantially eliminatedxe2x80x9d or xe2x80x9csubstantial ablationxe2x80x9d is meant a decrease in cell number such that less than approximately 50% of the cells remain viable, preferably less than approximately 20% of the cells remain viable, or more preferentially less than 10% of the cells remain viable.
By xe2x80x9csubstantially reduced viabilityxe2x80x9d is meant a decrease in the number of living cells in a population of cells relative to a control population such that less than approximately 50% of the cells remain viable, preferably less than approximately 20% of the cells remain viable, or more preferentially less than 10% of the cells remain viable.
By xe2x80x9cpre-cancerous tissuexe2x80x9d is meant mammalian tissue which is hyperplastic by histological examination and surgical-pathological evaluation well known to those skilled in the art of pathology.
By xe2x80x9ccancerous tissuexe2x80x9d is meant mammalian tissue which is no longer under the normal control of growth regulators. The cancerous nature of mammalian tissue is determined by surgical-pathological evaluation well known to those skilled in the art of pathology.
By xe2x80x9cantisensexe2x80x9d is meant nucleic acid wherein the noncoding strand of a target gene of interest is positioned for expression in a construct such that when expressed as MRNA, the single stranded antisense MRNA is complementary to and can hybridize to the MRNA of the target gene modulating its utilization. The sequence complementary to a sequence of the messenger RNA will usually be at least about 15 nucleotides, more usually at least about 20 nucleotides, preferably about 30 nucleotides or more, and more usually being fewer than 1000 nucleotides in length.
The particular site(s) to which the antisense sequence binds and the length of the antisense sequence will vary depending upon the degree of inhibition desired, the uniqueness of the sequence, the stability of the antisense sequence, or the like. Therefore, these factors will be determined empirically based on the experience obtained with a particular antisense sequence.
By xe2x80x9coperably attachedxe2x80x9d is meant a nucleic acid sequence to be expressed being fused at the 5xe2x80x2 end to a functional promoter element including transcriptional and translational (where applicable) initiation sites and being fused at the 3, end to a functional transcriptional and translational (where applicable) termination sequence.
By xe2x80x9cnucleic acid primerxe2x80x9d is meant a single stranded nucleic acid sequence, preferably DNA, that hybridizes to a nucleic acid sequence of interest for priming of DNA synthesis by DNA polymerase. The nucleic acid primers of the invention are designed for use in the polymerase chain reaction method and inverse polymerase chain reaction method (Shiramizu, B. et al. (1994), supra) and as described herein.
By xe2x80x9clabeled nucleic acid probexe2x80x9d is meant a single strand nucleic acid sequence to which is attached a label (such as a radiolabel; a biotinylation moiety; or other label known in the art and useful in Southern and Northern analyses). The nucleic acid sequence of the labeled probe used in the diagnostic method of the invention hybridizes to a genomic sequence near (i.e., within 5 kb, preferentially within 1 kb) a common HIV integration site. HIV integration at the site is associated with increased proliferation of the infected cell. An example of such a common integration site is the z exon of the fur gene where HIV integration is associated with macrophage proliferation (as described herein) or T-cell proliferation (Shiramizu, B. et al. (1994), supra)
By xe2x80x9ccell proliferationxe2x80x9d is meant increased cell growth in a test population above the rate of cell growth (e.g., cell division) of a control population such that cell growth is 20% above, preferably 50% above, or more preferably greater than 100% above a control population.
By xe2x80x9cgene associated with cell proliferationxe2x80x9d is meant a cell growth promoting gene (such as an oncogene) which when activated due to viral integration or other mutation results in increased cell proliferation. Cell proliferation genes include without limitation oncogenes such as c-fes/fps, ras, c-myc and others well known in the art; and genes encoding cytokines such as IL-6 and IL-10. An activated oncogene means an oncogene which increases the probability of the development of neoplasms (particularly malignant tumors) in a mammal (particularly a human). A gene associated with cell proliferation also includes a cell growth suppressing gene such that decreased gene expression of a suppressor gene results in cell proliferation.
By xe2x80x9cessentially non-functional mutated form of genexe2x80x9d is meant a gene or its gene product that is expressed or functions at 50%, preferably 20%, or more preferably less than 10% of wild type level.
By xe2x80x9cpreferential uptake by a macrophagexe2x80x9d is meant a macrophage able to incorporate an agent to a greater extent (20% greater, preferably 50% greater, or more preferably more than 100% greater extent) than cells of other cell types in the cell culture or in the mammal.
By xe2x80x9ckitxe2x80x9d is meant a combination of physical elements such as specific primers, labeled probes and other elements useful to practice the invention. The diagnostic kit of the invention comprises elements useful for the diagnosis of clonal macrophages in a mammalian tissue sample.
By xe2x80x9cmacrophagexe2x80x9d is meant a cell of the monocyte/macrophage lineage which is found in the spleen or has differentiated into a tissue macrophage. These cells include follicular dendritic cells (FDC), dendritic cells, Langerhans cells, as well as other tissue macrophages. The macrophage described herein is a non-tingible body macrophage not associated with scavenging cellular debris in the tissue.
By xe2x80x9cHIVxe2x80x9d is meant a human immunodeficiency virus of strains HIV-1, HIV-2, or other variants.
The methods of the invention and the kit of the invention are preferably designed for the diagnosis and treatment of clonal HIV- and non-HIV-associated macrophage-induced cancer in a human.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.